A hereditary immunoglobulin A abnormality: absence of light-heavy-chain assembly. Study of immunoglobulin synthesis in tonsillar cells.

A new immunoglobulin A abnormality, absence of assembly of alpha-chain and light-chain, was found in an adult female suffering from recurrent upper respiratory infection and tonsillitis since childhood, but otherwise healthy. The IgA abnormality was manifest in her serum by the presence of free alpha-chains, in her saliva by the presence of alpha-chains bound to secretory piece, and in her urine by the presence of free alpha-chains and free light-chains. The serum IgG and IgM were found to be complete, containing both heavy-chains and light-chains. Evidence for this immunoglobulin A abnormality was also found in the proposita's mother and elder son, demonstrating it to be a hereditary disorder. Studies performed with patient's tonsillar cells in short-term culture, using amino acids-(14)C, revealed synthesis and secretion of both free alpha-chains and free light-chains, in addition to synthesis and secretion of normally assembled IgG and IgM.

Expression of messenger RNA species coding for a Mr 43,000 peptide associated with ferritin in human leukemia-K562 cells and its down regulation during differentiation.

In the current study it was found that K-562 erythroleukemia cells express the superheavy (S) mRNA and the 43 Kd S peptide which were recently discovered in activated T-lymphocytes. The S mRNA was identified by its cross-hybridization with ferritin H complementary DNA probe and the S peptide (Mr 43,000) was identified by its immunoprecipitation with CM-H-9 monoclonal antibody specific for placental isoferritin. During terminal differentiation by hemin the level of S mRNA decreased below detection, with a concomitant diminution in the biosynthesis of the S peptide. In contrast, a significant increase in the level of ferritin light (L) and heavy (H) mRNAs was observed, resulting in an increase in the rate of biosynthesis of the H and L ferritin subunits. Removal of iron by desferrioxamine reduced the biosynthesis of ferritin L and H chains without altering the level of the corresponding mRNAs. Treatment with desferrioxamine did not affect either the level of S mRNA or the biosynthesis of the S peptide. This is a demonstration of a new mRNA species which is expressed in leukemic cells and is down-regulated during cell differentiation.

New monoclonal antibody enzymoassay for the specific measurement of placental ferritin isotype in hematologic malignancies.

A new enzymoimmunoassay, specific for the measurement of placental ferritin (PLF) isotype, has been described. Two monoclonal antibodies (McAbs) with different binding specificities to placental ferritin have been used in this assay. One antibody (CM-G-8) binds to all ferritins, whereas the second (CM-H-9) binds to placental ferritin only. In addition, a second enzymoassay was developed for the measurement of total common serum ferritin using CM-G-8 McAb. Serum levels of total ferritin and PLF were measured in healthy individuals and in patients with lymphoproliferative diseases and multiple myeloma. The majority of normal subjects were deficient in PLF in the serum. Increased serum levels of PLF were observed in patients with Hodgkin's lymphoma and non-Hodgkin's lymphoma of low and intermediate grades, as well as in patients with acute lymphocytic leukemia (ALL). Total ferritin was also elevated in these patients. Chronic lymphocytic leukemia (CLL) and multiple myeloma patients exhibited normal levels of common serum ferritin, whereas PLF in the serum was lacking.

Isoferritins in HIV infection: relation to clinical stage, CD8 lymphocyte binding and the pathogenesis of AIDS.

Placental isoferritins (PLF), known to be immunosuppressive in Hodgkin's disease and other states, were found to be increased in sera of subjects infected with HIV. We assayed for PLF using a 'sandwich' antigen capture enzyme-linked immunosorbent assay (ELISA) employing two monoclonal antibodies. Individuals with lymphadenopathy, with or without symptoms suggestive of AIDS-related complex, had the highest serum levels, which declined with progressive immunodeficiency. Total (normal) ferritins, in contrast, increased progressively with stage of disease. PLF was found on a subset of CD8 lymphocytes and appeared to block detection of the CD8 antigen by specific monoclonal antibodies. Elution of PLF by incubation with levamisole, but not by culture medium alone, led to the unblocking of the CD8 determinant on these cells. Profiles of isoferritins in HIV infection may provide clues to prognosis. PLF, a physiologic down-regulator of hematopoiesis and cellular immunity, could play a role in the progressive immune deficiency, marrow suppression and HIV expression that lead to AIDS.

Monoclonal antibody CM-H-9 detects circulating placental isoferritin in the serum of patients with visceral metastases of breast cancer.

The aim of the current pilot study was to determine whether placental isoferritin (PLF) can be detected in the serum of patients with metastatic breast cancer. Sera were obtained from breast cancer patients with metastatic disease (n = 100), from breast cancer with no evidence of disease (n = 70) and from healthy female controls (n = 34). PLF and total serum ferritin levels were independently measured using specific monoclonal antibody ELISAs in a double-blind study. It was found that the mean serum PLF levels were significantly elevated only in patients with visceral metastases (lung, liver, brain) compared with the levels of patients with non-visceral metastases (bone, skin) or with healthy controls. Contrary to this, analysis of total serum ferritin levels did not reveal significant differences between these groups. Considering 0-10 units/ml as a PLF negative result, it was found that PLF was negative in 87.5% of healthy controls and in 96% of breast cancer patients with no evidence of disease. In contrast, PLF was positive in 73% of the patients with visceral metastases and in 29.7% of those with non-visceral metastases. The striking difference between visceral and non-visceral metastases is not yet understood. It could result from a difference in the degree of vascularisation or, alternatively, a difference in the cell types and genes expressed by cells metastasizing to visceral or non-visceral organs.

Alterations of cell surface antigens induced by placental isoform of ferritin in human carcinoma cell lines.

AIM: the ability of the breast cancer-associated placental acid isoform of ferritin (p43-PLF) to modulate cell surface expression of HLA, CD59 (protectin) and CD66 antigen (adhesion antigen related to CEA) was examined. METHODS: the expression of these antigens in human breast carcinoma cell lines BT-20, T47D and MDA-MB-468 was determined with the aid of flow cytometry and monoclonal antibodies. RESULTS: PLF induced a transient up-regulation followed by a down regulation of cell surface protectin (CD59 antigen) on the cell surface of T47D and to a lesser extent, BT-20 human breast carcinoma cell lines. Furthermore, PLF down-regulated cell surface expression of CEA-related CD66 antigen on both these cell lines. No PLF-induced alterations of protectin, CD66 antigen and HLA class I antigen were found on the MDA-MB-468 breast cancer cell line. CONCLUSIONS: breast cancer-associated p43 induces alterations of the expression of cell surface molecules in breast cancer cells which could have an effect on the modulation of cancer cell adhesive interactions.

Downregulation of lymphocyte mitogenesis by breast cancer-associated p43.

Placental isoferritin-associated p43 has proven to induce immune suppression during pregnancy in order to avoid rejection of the fetus' alloantigens by maternal lymphocytes. It has been demonstrated previously that p43 is also synthesized by breast cancer cells and can also be found on the surface of a subpopulation of T cells in women with this disease. Therefore, it was the aim of the present study to investigate if breast cancer-associated p43 has immunosuppressive properties. In 40 women undergoing surgical excision of a suspicious lump in their breast, blood was withdrawn and lymphocytes were isolated. Lymphocyte cultures were incubated with p43 antigen and anti-p43 antibody (CM-H-9). In a second series, lymphocyte mitogenesis was activated by addition of concanavalin A (Con A), Con A + p43 and Con A + anti-p43, respectively. While lymphocytes of breast cancer patients (n = 21) and women with benign breast disease (n = 19) incubated with p43 as well as with anti-p43 antibody did not show any difference in terms of incorporation of [3H]thymidine, activation of lymphocytes by addition of Con A was significantly inhibited after addition of p43 antigen in breast cancer patients compared to women with benign breast disease (P = 0.0178). Analysis of prognostic factors for breast cancer showed that inhibition of lymphocyte mitogenesis was dependent on the degree of tumor differentiation and was significantly higher in well differentiated tumors (GI) compared with more dedifferentiated tumors (GIII). The present study shows that breast cancer-associated antigen p43 is able to induce immune suppression in breast cancer patients but not in women with benign breast disease.

Expression of p43 in breast cancer tissue, correlation with prognostic parameters.

Placental isoferritin (PLF) and its unique superheavy chain p43 have been recently described as being synthesized by breast cancer cell lines but not by normal breast epithelial cells. Since previous reports have demonstrated a correlation between the content of 'normal' ferritin in breast cancer tissue and the degree of differentiation and prognosis, we have determined p43 in the cytosol of 122 breast cancer samples by use of the new monoclonal antibody CM-H-9. The synthesis of p43 showed a significantly negative correlation with tumor size (P = 0.0001), histologic grading (P = 0.0038), nuclear pleomorphism (P = 0.0019), rate of mitosis (P = 0.0002), lymphocytic reaction (P = 0.0001) and a significantly direct correlation with estrogen receptor status (P = 0.0009). Although patients with a higher p43 content showed a trend for a better outcome (median follow-up: 61.4 months), an independent influence of the cytosolic p43 content on survival could not be confirmed by a multiple Cox model. Therefore it seems that p43's prognostic impact is linked to the highly significant correlation with features of differentiation although a statistical bias in the Cox model due to the limited number of patients must also be taken into account. On the other hand, the significant correlation of p43 expression with factors for good prognosis was striking and consistent and warrants further research of this tumor product.

Activated lymphocytes from breast cancer patients express the characteristics of type 2 helper cells--a possible role for breast cancer-associated p43.

P43, a breast cancer-associated antigen, has been repeatedly described as an immunosuppressive factor. The objective of the present study was to investigate whether immune dysregulation induced by p43 affects the profile of cytokines secreted by mitogen-stimulated lymphocytes in breast cancer patients as compared with stimulated lymphocytes in women with benign tumors. The study consisted of 32 women undergoing surgical excision for a suspicious lesion in their breast. Histology revealed malignant breast disease in 20 patients and benign lesions in 10 patients. Lymphocytes isolated from peripheral blood were activated by Conconavalin A (Con A) with and without the addition of p43 and the concentrations of cytokines (IL-2, TNF-alpha, IFN-gamma, IL-4, IL-10 and IL-6) secreted into the culture medium were determined. Lymphocytes of patients with malignant breast disease stimulated with Con A secreted a significantly higher concentration of IL-10 compared with lymphocytes of patients with benign tumors. No significant differences were found between the two groups regarding the levels of IL-2, TNF-alpha, IFN-gamma and IL-4. Cytokine concentrations were analyzed according to the type 1/type 2 cytokine profile (IL-2, TNF and IFN-gamma and IL-4, IL-6 and IL-10, respectively). This analysis revealed no significant differences in IL-2, TNF or IFN-gamma between benign and malignant tumors. However, in the type 2 cytokines, lymphocytes from cancer patients secreted significantly higher levels of IL-4 (27.3 +/- 7.2 U/ml) and IL-10 (44.1 +/- 22.3 U/ml) than did the lymphocytes from patients with benign disease (21.4 +/- 7.3 and 1.8 +/- 0.3 U/ml, respectively). The addition of p43 to the culture medium significantly enhanced the levels of IL-4 secreted by lymphocytes in both groups of patients (malignant disease, from 27.3 +/- 9.2 to 40.7 +/- 6.3 U/ml; benign disease, from 21.4 +/- 7.3 to 28.4 +/- 2.1 U/ml). P43 antigen significantly enhanced the low levels of IL-10 in the benign lymphocytes (from 1.8 +/- 0.4 to 8.4 +/- 1.5 U/ml) while the high levels of IL-10 secreted by the PBL in patients with malignant tumors were not significantly increased (44.1 +/- 22.3 versus 50.1 +/- 12.6 U/ml). The study showed a difference in the immune response of lymphocytes between malignant and benign tumors. When the current results were analyzed according to the type of response, i.e. in terms of whether at least two cytokines of either type 1 or type 2 were elevated, a significant type 2 response was observed in the PBL of patients with malignant breast cancer (IL-10 and IL-4). These results may explain why antitumor response is impaired in patients with breast cancer.

Placental isoferritin levels during first trimester of normal and complete molar gestations.

Serum placental isoferritin levels (PLF) levels were measured in 33 patients admitted for routine scanning of a first trimester normal singleton pregnancy and six patients who were hospitalized for uterine evacuation of a complete molar gestation. Venous blood was obtained upon admission and before curettage, when necessary. Serum was separated into glass tubes, immediately frozen and stored at -20 degrees C until analysed. The mean serum PLF levels were 18.1 (+/- 14) U/ml and 5.5 (+/- 2) U/ml for normal and molar gestations, respectively, with a significant difference between the two groups (P = 0.001). Sixty-seven per cent of normal pregnancies had serum PLF levels > or = 10 U/ml, whereas none of the molar gestation group reached this threshold level. Furthermore, the molar gestation group's low serum PLF levels remained unchanged throughout the entire follow-up period and until their beta-human chorionic gonadotrophin levels were undetectable. Unlike normal pregnancies, the molar trophoblast does not seem to secrete or synthesize PLF, suggesting that the complete molar placenta has different protein-producing capabilities when compared with those found in normal pregnancies. Further studies, including serum PLF among other categories of gestational trophoblastic neoplasms, are recommended before this data can be integrated into routine clinical work.

Placental isoferritin as a marker of early abortion in pregnancies induced by in vitro fertilization.

The relatively high rate of early pregnancy loss in artificial reproductive technology-induced conceptions has driven researchers to seek for an efficient diagnostic tool for estimating the gestational risk in these cases. Monitoring early gestation normalcy using serial beta-human chorionic gonadotrophin (beta hCG) measurements requires several days before diagnosis can be established. The objective of this study was to determine whether placental isoferritin (PLF) can be used as a predictive marker of normal pregnancy development during early stages of in vitro fertilization (IVF)-induced pregnancy. Ninety-three consecutive IVF cycles were investigated. Blood samples for PLF (enzyme linked immunosorbent assay; ELISA) and beta hCG (radio-immunoassay; RIA) determination were obtained from all women on days 11, 13 and 15 following embryo transfer. Placental isoferritin was detectable in the serum 11 days after embryo transfer in IVF conception cycles. These levels were significantly higher in normally developing pregnancies (n = 18) than in cases which eventually aborted spontaneously (n = 9) during the first trimester (mean +/- s.d.; 33 +/- 28 U/mL as compared with 1 +/- 2 U/mL; P = 0.0004; Wilcoxon test; sensitivity 94.5 per cent, specificity 88.9 per cent, positive predictive value 89.9 per cent, negative predictive value 94.5 per cent). Moreover, in those patients who later aborted, lower than normal PLF levels were detected long before the decline in beta hCG titres was evident. Considering its suppressor activity, it is expected that PLF levels would be high at the initiation of normal pregnancy. This may explain the present finding of low PLF levels in abnormally developing IVF-induced pregnancies. These preliminary data suggest that PLF can be used as a sensitive marker for detecting those cases destined to abort at a very early stage. However, further studies are still required on spontaneous conceptions, before this test can be recommended for routine clinical application.

Diminished expression of placental isoferritin p43 component in first trimester abnormal pregnancies.

Human placental isoferritin (PLF) is a sub-type of human ferritin mainly composed of a 43 kD protein, which has an immunosuppressive activity and may be involved in the downregulation of the maternal immune system during pregnancy. The aim of this study was to evaluate the distribution of p43 in the placental tissue of abnormal first trimester pregnancies. Samples of villous and decidual tissues were collected between 7 and 12 weeks' gestation from 28 missed abortions and eight complete moles. Samples of placental tissue from 20 normal pregnancies of similar gestational age were used as controls. The localization of p43 was determined by immunohistochemical techniques using CM-H9 monoclonal antibody. Compared to controls, specific p43 immunoreactivity was low in the villous syncytiotrophoblast of missed abortions and absent from all villous cellular types in complete moles. These findings correlate well with the low level of maternal serum PLF found previously in early pregnancy failures and molar gestation. This suggests that PLF may be involved in the pathogenesis of early pregnancy disorders related to an abnormal placentation.

Placental isoferritin measured by a specific monoclonal antibody as a predictive marker for preterm contraction outcome.

The serum levels of placental isoferritin and normal ferritin in 25 women with preterm contractions (mean +/- SE gestational age 28.8 +/- 4.7 weeks) were compared with those in 14 control women with uncomplicated pregnancies (29.1 +/- 7.3 weeks). The serum concentration of placental isoferritin in women with preterm contractions (15.3 +/- 6.2 U/mL) was significantly lower than that in normal pregnant women (87.6 +/- 22.6 U/mL) (P = .005). The level of normal ferritin in women with preterm contractions (30.6 +/- 4.16 ng/ml) was lower than that in women with normal pregnancies (67 +/- 9.6 ng/mL) (P = .01); however, both were within the normal range. Serum placental isoferritin levels correlated with pregnancy outcome; low placental isoferritin (up to 10 U/mL) was a sensitive (71.5%) indicator of preterm labor. Low placental isoferritin had a positive predictive value of 59% and a negative predictive value of 71%. These results suggest that placental isoferritin may serve as a predictive marker for the prognosis of preterm contractions.

Preparation and characterization of monoclonal antibodies specific to placenta ferritin.

Ferritins are a group of isomeric proteins which function as the major iron-storage protein of mammalian tissues. Some of the isoferritins have low isoelectric points, and are found in placenta and in malignant tissues and have therefore been termed carcinofetal ferritins. With the use of hybridoma technology, monoclonal antibodies (McAbs) specific to human placenta ferritin(s) were produced in order to characterize the heterogeneity of the molecule and to answer the question whether a specific antigenic determinant is associated with placenta and/or carcinofetal ferritin(s). Two McAbs designated H-9 and G-8 were developed. McAb H-9 bound specifically and exclusively to the ferritin isolated from human placenta, whereas G-8 McAb bound to placenta ferritin and cross-reacted with ferritins isolated from human spleen and liver. No cross-reaction was observed between H-9 and G-8 reactive determinants. It was found further that the two antigenic determinants - the one recognized by G-8 and that recognized by H-9 McAbs - are molecularly associated on placenta ferritin. The results of this study led us to term the G-8 a 'common' ferritin antigenic determinant and H-9 a 'private' embryonic ferritin determinant.

The regulatory role of adenosine activated T-lymphocyte subset on the immune response in humans. II. Adenosine induced expression of T8 antigen.

Adenosine sensitive suppressor T cells (ESen) were isolated from healthy donors and continuous cultures of these cells were established. It was found that the continuous proliferation of ESen cells required IL-2, Adenosine and ConA. The reactivity of ESen cells with OKT3, OKT4 and OKT8 monoclonal antibodies was studied before and after incubation with adenosine. The majority of freshly isolated ESen cells were OKT8+ cells. During culture, the majority of the proliferating ESen cells were OKT4; however, following a brief incubation of cultured ESen cells with adenosine, an increase in OKT8+ cells and a decrease in OKT4+ cells were observed. A fraction of ESen cells exhibited simultaneously OKT4+ and OKT8+ antigens similar to thymocytes. Furthermore, adenosine treatment of thymocytes resulted in a small increase in OKT8+ and OKT3+ cells and a parallel decrease in OKT4+ cells. These results indicate that adenosine may play a regulatory role in the differentiation of lymphocytes programmed to carry out a specific function.

Elevated serum ferritin level in acute myocardial infarction.

Serum ferritin level was determined in 20 patients with acute myocardial infarction (AMI) during the first 10 days post infarction. Starting on the second day, a gradual increase in serum ferritin level was detected, reaching a maximum of four times the initial level on the sixth day after the infarction. In addition, a significant increase in ferritin content was found in the peripheral blood monocytes on the fifth day after the event. The control group comprised six patients suffering from chest pains not due to AMI. In all of them the serum ferritin level was found to be within normal limits. Peripheral blood monocytes derived from healthy individuals incubated with hydrocortisone, showed a significant enhancement of their ferritin content, a finding suggesting that these cells activated by steroids during stress could be a source of the increased serum ferritin level following AMI. It is concluded that measurement of serum ferritin may be used as a complementary tool for confirming the diagnosis of AMI.

FBL blood test as a predictive marker of breast cancer in high risk women.

The potential use of the ferritin bearing lymphocytes (FBL) blood test which enumerates oncofetal FBL as a biomarker for early breast cancer was explored. Analysis of the FBL positive test results carried out on high risk women who underwent biopsy in 1983-84 was found to be a significant predictor for early breast cancer (relative risk (RR) = 2.9, 95% confidence interval (CI) 1.4-5.8). A group of 635 women from the above study diagnosed in 1983-84 as having no evidence of breast cancer was further traced 8 years later with the aid of the National Cancer Registry. We identified 35 malignancies, including 19 cases of breast cancer. The RR of breast cancer for the FBL positive group was 2.51; 95% CI = 1.04-6.07 while for the other malignancies it was 0.93, 95% CI = 0.30-2.84. All the breast cancer cases in the FBL positive group were of the infiltrative duct carcinoma category and 71% were in early stage. In contrast, in the FBL negative group, 60% of the cases were infiltrative ductal carcinoma and most of them were at stage III. Positive FBL is associated with early manifestation of breast cancer and may be considered as a tool for the screening of breast cancer in high risk women.

Placental type isoferritins in chronic liver diseases.

Serum ferritin levels in chronic liver diseases were measured by an enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies (McAb) against placental ferritin. One of the McAbs (CM-H9) recognizes a specific placental-like isoferritin (PLF) only, while the other McAb (CM-G8) recognizes an isoform (CF) common to human placenta, liver and spleen. The different diseases studied were primary biliary cirrhosis (PBC-14 patients), chronic active hepatitis (CAH-12 patients), alcoholic cirrhosis (AC-18 patients) and cryptogenic cirrhosis (CRY-26 patients). Increased levels of both isoferritine were found in all these liver disorders, with an overall incidence of 80% for CF and 40% for PLF. The mean level of CF was significantly above normal in PBC, AC and CRY and that of PLF in PBC and CRY. Circulating placental-type isoferritins have not been previously described in patients with liver disorders. Our findings indicate that elevation of serum ferritin in liver diseases is caused by different isoferritin components.

[Placental isoferritin (PLF): early predictor of fetal growth retardation]. / Früherkennung fetaler Wachstumsretardierung durch Bestimmung des plazentaren Isoferritins.

OBJECTIVE: Maternal immunoreaction against the embryo can be responsible for fetal growth retardation. The secretion of PLF during pregnancy could be reliable for a diminished immunoreaction of maternal lymphocytes against the embryo. The aim of the present study was to elucidate a possible correlation between PLF and fetal growth retardation. METHODS: In this study, blood samples of 402 pregnant women were obtained between the 12th and 16th weeks of gestation. The serum levels of PLF were compared with birth weight, percentile of birth weight, and gestational age. RESULTS: Women with children whose birth weight was below or equal to the 10th percentile for gestational age showed significantly lower PLF levels (11.4 U/ml, n = 107) as compared with women whose newborns were of normal weight (19.9 U/ml, n = 295; p < 0.004). CONCLUSION: Determination of the PLF level could serve to identify women at risk of having growth-retarded babies.